BAS - Institute of Botany

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Background

Aims & Objectives

Activities

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Background

Bulgaria is characterized by its rich and diverse flora. Over 120 plant medicines are been manufactured in Bulgaria. One of the main factors that jeopardize natural resources of medicinal plants is intensive extirpation for industrial purposes and herb gathering for export of crude drug. Protection measures have been applied to preserve rare and threatened medicinal plants for further rational use. According to the new Medicinal Plants Act (2000), 743 species of the Bulgarian flora come under these law regulations and need real strategies for in situ and ex situ conservation. New ways of increasing or compensation of the limited supply of some rare and threatened plant species but economically valuable have to be developed.

Summer snowflake (Leucojum aestivum L.) contains the important alkaloid galanthamine (Gal), with strong anticholinesterase activity, in highest concentration (compared with the other Gal sources: Narcissus and Galanthus). This active substance is currently used in treatment of numerous nervous diseases of the central and peripheral nervous system including Alzheimer’s syndrome. Leucojum herbage gathered in the wild is the natural Gal source for the Bulgarian patented medicines Nivalin, Nivalet, Nivalin P and Nivatonin, as Sopharma Ltd. is the exclusive producer. In Bulgaria, large natural populations of L. aestivum have been exploited since 1960. Overutilisation of populations and destruction of habitats led to protection measures. Large areas of ecosystem complexes of longos type were proclaimed nature reserves: Kamchia, Baltata, Arkoutino, Gorna and Dolna Topchia. In 1970 the Ministry of Forests and Forestry gave the statute of protected areas to 11 localities. The species were included in the Red Data Book of Bulgaria in the category “endangered”. Since 1981 harvest is allowed upon annual authorization from the Interdepartmental Commission of Snowflake and from the Ministry of Environment and Waters, after estimation of the population status and quota determination.

Supplies of L. aestivum are currently insufficient to meet the increasing pharmaceutical demand. As a consequence, fluctuations and overall decrease in Gal production have occurred, whereas the market for Gal is increasing. If large-scale extraction and characterization for industrial purposes have been developed, no industrial plantation of snowflake is been used at the moment in Bulgaria.

For decades, biotechnological approaches have been developed in several cases to answer the demand with nature-identical active substances in an environment-friendly manner. In addition, numerous in vitro culture studies have been performed in Amaryllidaceae for micropropagation purposes. Today, however, these species are still considered as recalcitrant. In vitro culture of Leucojum has been initiated as well but less information is currently available. If micropropagation is possible and slow-growing calli were obtained, to our knowledge there is no report mentioning Gal measurements in Leucojum in vitro cultures.
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Aims & Objectives

The aim of our Project is to develop alternative approaches for bioproduction of Gal, thus ensuring both: supply of the pharmaceutical company Sopharma with row material and protection of the natural Bulgarian populations of L. aestivum. These approaches are based either on biotechnological systems (bioreactor cultivation and in vitro multiplication) or on agronomical/horticultural practices (clonal selection, rapid multiplication, environmental conditioning).

Main project objectives are: to evaluate the current status of the Bulgarian populations of L. aestivum; to constitute representative germplasm collection from wild populations; to evaluate snowflake agronomic and biochemical performances of L. aestivum under field conditions; to characterize soil composition of diverse natural L. aestivum populations; to evaluate and to select clones with high contents of biologically active substances for further multiplication; to identify suitable biological material for in vitro experiments, to develop efficient in vitro cell cultures and micropropagation; to characterize alkaloid production in vitro and in vivo; to develop lab scale in vitro cultures producing biologically active substances.

Activities

Task 1: Reorganisation of the labs & standardisation (All Partners)
Leader: M. Stanilova

to get the necessary equipment (Partners 1 & 2);

to set the new Biotechnological Laboratory of Medicinal Plants (Partner 1);

to standardize lab procedures (all Partners).

Task 2. Germplasm gathering from wild populations (Partners 1, 4)
Leader: Ch. Gussev

to assess natural populations of L. aestivum, according their status;

to select the most appropriate populations for gathering of plants and bulbs;

to collect plant material for in vitro and in vivo cultivation ;

to create a data base on current diversity of Leucojum populations and habitats.

Task 3. Field cultivation and selection (Partner 1)
Leader: T. Stoeva
Sub task 3.1. In vivo cultivation of L. aestivum and multiplication

to reorganise the experimental field plot and facilities;

to maintain plant material for in vitro cultivation;

to optimise twin-scaling method for rapid bulb multiplication and to propagate clones.

Sub task 3.2. Effects of different parameters on biomass productivity and alkaloid content

to analyse soil samples gathered from different localities;

to study the phenophase dynamics of alkaloids/Gal accumulation and assimilation of nutrient elements;

to assess biomass productivity and Gal content among individuals from different populations.

Sub task 3.3. Clonal selection of L. aestivum and multiplication of elite clones

to set up a nursery garden (collection) for the selection of elite clones;

to improve the genetic background by clonal selection.

Task 4. In vitro cultivation (Partners 1, 2 & 4)
Leaders: M. Ilieva-Stoilova & M. Stanilova

to evaluate biotechnological approaches for alkaloid bioproduction;

to determine the main bottlenecks hampering the in vitro approaches;

to propose ways of solving them.

Sub task 4.1. Callogenesis (Partner 2)

to investigate callus formation in L. aestivum;

to establish friable callus culture and to optimise callus subcultivation;

to compare callogenesis from different Leucojum populations.

Sub task 4.2. Micropropagation (Partners 1, 4)

to investigate direct and indirect morphogenesis in L. aestivum;

to obtain shoot-clumps and to establish liquid shoot-clump culture from L. aestivum;

to regenerate in vitro whole plants and to adapt them to unsterile conditions;

to evaluate stability of in vitro plantlets by karyological means and DNA measurements.

Sub task 4.3. Liquid cultures of L. aestivum (Partner 2)

to obtain suspension culture from L. aestivum;

to study growth conditions and alkaloid production in parallel;

to proceed scaling-up in a lab 3L-bioreactor.

Task 5. Alkaloid determination (All Partners)
Leader: S. Yanev

to develop extraction and analytical methods for alkaloid determinations (both qualitative and quantitative) in plants, in vitro shoots, cell cultures, shoot clumps and calli;

to perform routine alkaloid analysis on all biomasses during the project.

Sub task 5.1. Methodologies for alkaloid extraction, identification and quantification

to develop a semi-quantitative fast TLC screening method for Gal-assessment in the field (Partner 1);

to develop more accurate analytical method (HPLC) for Gal and alkaloid identification;

to confirm the TLC results by HPLC analysis

to establish Gal CPC (Partner 4).

Sub task 5.2. Screening of germplasm
Sub tasks 5.3: Alkaloid content in vivo
Sub tasks 5.4.: Alkaloid content in vitro

Task 6. Training of young scientists (All Partners)
Leader: M. Burrus Top